The epidermal cornified cell envelope (CE) is a complex protein/lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin, i.e. its ability to prevent the loss of water and ions and to protect from environmental hazards. The phenotypic consequence of failing to form a functional epidermal barrier is dramatically illustrated by the p63 knockout mouse which is born alive, but dies within hours due to dehydration (Project 1). The major protein approaches has been used by our laboratory to determine the phenotypic consequences of both loss-of- function and gain-of-function mutations in the loricrin gene. Surprisingly, loss of loricrin expression is well tolerated due to a backup system that compensates for its loss. This compensatory responses causes the up- regulation of several known CE components and this inductive event occurs in utero. Preliminary data documenting that novel CE components are induced during this compensatory response are also included. This project will characterize these recently discovered genes and attempt to identify other CE components expressed in the absence of loricrin (Specific Aim 1). Another goal of this project is to analyze gene regulatory mechanisms that allow mutant embryos to compensate for the loss of loricrin (Specific Aim 2). The knowledge of these mechanisms will not only elucidate the molecular events that lead to phenotypic compensation in loricrin knockout mice, but may also identify targets for therapeutic intervention in cases of barrier defects in humans (e.g., in premature infants). Transgenic mice have also been generated which express a gain-of-function loricrin mutation identical to that responsible for inherited skin disorders in man. This mutation causes a defect in epidermal barrier function, but surprisingly does not require a direct interaction with wild-type loricrin, nor incorporation into the CE. Instead, the mutant protein accumulates in the nucleus. A series of experiments are proposed to further elucidate the mechanisms by which this loricrin mutation impairs epidermal barrier function and causes inherited skin disorders (Specific Aim 3).